JP4338527B2 - Oligomer compounds that regulate HIF-1α expression - Google Patents

Description

(Related application)
This patent application is a continuation application of US Provisional Application No. 60 / 370,126, filed April 5, 2002, the entire text of which is incorporated herein by reference.

  The present invention provides compositions and methods for modulating HIF-1α expression. In particular, the present invention relates to oligomeric compounds, and preferred compounds include oligonucleotides, which can specifically hybridize with a nucleic acid encoding HIF-1α. This oligonucleotide compound has been shown to modulate the expression of HIF-1α and its pharmaceutical formulation and use for the treatment of cancer and pre-eclampsia is disclosed.

  In order for a solid cancer to grow to a few millimeters or more, it is necessary to establish a blood supply and enhance glucose metabolism. Tumor organisms how cancer perceives hypoxia and how it responds by activating hypoxia-inducible genes and selecting angiogenic factors to establish the blood system It is a central subject of science. Many tumors contain a hypoxic microenvironment, which is associated with malignancy, metastasis, and resistance to radiation therapy and chemotherapy.

  The discovery of hypoxia-inducible factor-1 (HIF-1) gave some insight into the regulation of hypoxia-inducible genes (US Pat. No. 5,882,914 and International Publication No. WO96). / 39426 and WO99 / 48916). HIF-1 is composed of two subunits, HIF-1α and HIF-1β, such as angiogenic factors such as VEGF and glycolytic enzymes and glucose transporters 1 or 3 (GLU-1 or 3) Binds to hypoxia-responsive components (HREs) in the enhancer of the gene encoding glycolysis-related proteins.

  It has been shown that by introducing an antisense HIF-1α plasmid into a tumor and incorporating the negative control of HIF-1α, VEGF production is suppressed and the microvessel density in the tumor is reduced (International Publication No. 1). WO 00/76497; Sun X et al., Gene Therapy (2001) 8, 638-645). The plasmid contained a 320-bp cDNA fragment encoding the 5'-end of HIF-1α (nucleotides 152-454; Genebank AF003698). Furthermore, the above international application describes a method based on the fact that expression vectors should be used in conjunction with immunotherapeutic agents. However, the main weakness of this expression plasmid approach is that it is not suitable as a therapeutic because of the size of the plasmid and the nucleolytic enzyme sensitivity of the expression product.

  In addition to HIF-1α fragment expression plasmids, a few antisense oligonucleotides targeted to HIF-1α have been designed as tools to study specific biological mechanisms or targets. For example, inhibition of HIF-1α expression in explants under hypoxia with antisense has been shown to inhibit TGFβ expression (Caniggia, I. et al., J. of Clinical Investigation, March 2000, 105). , 577-587). In this particular study, only one antisense oligonucleotide was synthesized, which was a phosphorothioate targeting the sequence adjacent to the start codon AUG of HIF-1α mRNA. Its sequence is HIF-1α 5'-GCCGGGCGCCTCTCCAT-3 ', and negative regulation of HIF-1α was revealed at the mRNA level. This oligo has been used to study the role of HIF-1α in hyperchorionic trophoblast development and invasion and has been linked to the potential role of HIF-1α in preeclampsia (Caniggia, I. et al. Et al., Placenta (2000), 21, Supplement A, Trophoblast Research 14, S25-S30).

  Other studies using the same oligonucleotide sequences described above showed that antisense inhibition of HIF-1α decreased peroxisomal proliferative active receptors (PPARs) (Narravula, S. and Colgan, S.P.). , J. of Immunology, 166, 7543-7548 (2001)). The above oligos were also used to show that nickel is required for induction of plasminogen activator inhibitor-1 (PAI-1) of HIF-1α (Andrew, AS, Klei LR). , Barchowsky, A., Am. J. Physiol.Lung Cell MoI. Physiol., 281, L607-L615 (2001)).

Single antisense oligonucleotides have also been used in the study of two splicing variants of the hypoxia-inducing factor HIF-1α as potential partners for neuronal ARNT2 dimers. The antisense oligonucleotide was a phosphorothioate modification of the 5′-TCTCTCCGTTCTCGCC-3 ′ sequence. Treatment of the cells with this oligonucleotide suppressed [ ³ H] thymidine incorporation, but had no apoptotic effect in normoxic cells (Drutel et al., Eur. J. Neurosci., 12, 3701-3708 ( 2000)).

  In addition, a single antisense oligonucleotide against HIF-1α has been shown to inhibit gene expression of cardiac endothelin (ET) -1, and HIF-1α expresses intramyocardial ET-1 gene in cardiac disorders It was hypothesized that it was involved in enhancement (Kakinuma, Y. et al., Circulation, 103, 2387-2394 (2001)). The antisense oligonucleotide had the following sequence: CCTCCATGGGCGAATCGGTGC.

  Currently, there is no known therapeutic antisense agent that effectively inhibits the synthesis of HIF-1α and can be used for disease treatment. Therefore, a drug that effectively inhibits the function of HIF-1α and is used, for example, for the treatment of cancer and preeclampsia is desired.

(Summary of Invention)
The present invention is directed to oligomeric compounds, particularly LNA antisense oligonucleotides, which target nucleic acids encoding HIF-1α and modulate HIF-1α expression. Also provided are pharmaceuticals and other compositions containing the oligomeric compounds of the present invention. Further provided is a method of modulating HIF-1α expression in a cell or tissue comprising contacting the cell or tissue with one or more of the oligomeric compounds or compositions of the invention. Also, animals that have or tend to have a disease or condition associated with the expression of HIF-1α by administering one or more therapeutically or effective amounts of the oligomeric compounds or compositions of the present invention. Alternatively, a method for treating a human is also disclosed. Further provided are methods of using oligomeric compounds for inhibiting expression of HIF-1α and treating diseases associated with these HIF-1α. Examples of such diseases include different types of cancers, particularly common and primary breast cancers such as primary and metastatic breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, other gastrointestinal cancer, lung cancer, cervical cancer, ovary Cancer and brain tumors, as well as pre-eclampsia, inflammatory visceral diseases and Alzheimer's disease are mentioned as well. Examples of other cancers include colon cancer, liver cancer, thymic cancer, kidney cancer, testicular tumor, stomach cancer, small intestine cancer, intestinal cancer, esophageal cancer, spinal cord cancer, sinus cancer, bladder cancer or urinary tract cancer. .

(Definition)
As used herein, the term “target nucleic acid” refers to DNA encoding hypoxia-inducing factor or hypoxia-inducing factor-1α (HIF-1α), RNA translated from such DNA (pre- mRNA and mRNA), and cDNA derived from such RNA.

  As used herein, the term “gene” refers to exons, introns, genes including non-coding 5 ′ and 3 ′ end regions and regulatory regions, and all currently known variants and future elucidations. Means any variant.

  As used herein, the term “oligomer compound” includes, for example, binding to target genes “chimeric plasts” and “TFO” by hydrogen bonding, target genes “antisense inhibitors”, “siRNA”, Desirable for humans through binding of “ribozymes” and “oligozymes” to RNA transcripts or binding to proteins encoded by the target genes “aptamers”, “Spiegelmers” or “decoys” An oligonucleotide that can induce a therapeutic effect.

  As used herein, the term “mRNA” refers to any currently known mRNA transcript of a target gene, as well as any transcript that can be identified.

  The term “modulation” as used herein means either an increase (stimulation) or a decrease (inhibition) of gene expression. In the present invention, inhibition is a preferred form of regulation of gene expression and mRNA is a preferred target.

  As used herein, an antisense compound “targets” a particular target nucleic acid means that an antisense oligonucleotide is attached to a cell, animal or so that the antisense compound binds and modulates the function of that target. Means to give to humans.

  The term “hybridization” as used herein refers to hydrogen bonding between complementary nucleoside or nucleotide bases, such as Watson-Crick, Hoogsteen, reverse Hoogsteen hydrogen bonding. Watson and Crick about 50 years ago, deoxyribonucleic acid (DNA) consists of double strands, which are held together by a helix structure formed by hydrogen bonds formed between opposing complementary nucleobases. I showed that it fits. The four nucleobases commonly found in DNA are guanine (G), adenine (A), thymine (T) and cytosine (C), of which nucleobase G pairs with C and nucleobase A Is paired with T. In RNA, the nucleobase thymine is replaced by the nucleobase uracil (U) and pairs with A in the same way as the nucleobase T. The chemical group in the nucleobase involved in standard double helix formation constitutes the Watson-Crick face. Two years later, Hoogsteen has a Hoogsteen surface in which purine nucleobases (G and A) can be identified from the outside of the double helix in addition to the Watson-Crick surface, and pyrimidine nucleic acid via hydrogen bonding. It has been shown that triple helix structures are formed when used to bind bases.

  In the context of the present invention, “complementary” refers to the ability to make an exact pair between two nucleotide or nucleoside sequences. For example, if one nucleotide at a position of an oligonucleotide can hydrogen bond with the nucleotide at the corresponding position of DNA or RNA, the oligonucleotide and DNA or RNA are considered complementary to each other at that position. DNA or RNA and oligonucleotides are considered to be complementary to each other because a sufficient number of nucleotides in the oligonucleotide form hydrogen bonds with the corresponding nucleotides in the target DNA or RNA to form a stable complex (complex ) Can be formed. In order to be stable in vivo or in vitro, the sequence of the antisense compound need not be 100% complementary to its target nucleic acid. Thus, the terms “complementary” and “specific hybridization” refer to the normality of a target without the antisense compound specifically binding with sufficient strength to the target compound and affecting the function of the non-target mRNA. Means disturbing the desired function as desired.

  The term “nucleic acid analog” refers to a compound that binds a non-natural nucleic acid. For nucleic acid analogs, see, for example, Freier & Altmann (Nucl. Acid Res., 25, 4429-4443 (1997)) and Uhlmann (Curr. Opinion in Drug & Development, 3 (2), 293-213 (2000)). There is a report. Scheme 1 shows a selected example.

  The term “LNA” refers to an oligonucleotide containing one or more bicyclic nucleoside analogs, also referred to as an LNA monomer. As for the LNA monomer, International Publication No. WO99 / 14226 and subsequent International Publication Nos. WO00 / 56746, WO00 / 56748, WO00 / 66604, WO00 / 125248, WO02 / No. 28875, WO 02/094250 and International Application No. PCT / DK02 / 00488. The thymidine LNA monomer mentioned as a specific example is (1S, 3R, 4R, 7S) -7-hydroxy-1-hydroxymethyl-5-methyl-3- (thymin-1-yl) -2,5-dioxa- Bicyclo [2: 2: 1] heptane.

  The term “oligonucleotide” in the context of the present invention is preferably an oligomer (also called an oligo) or a nucleic acid polymer (eg ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) or a known nucleic acid analogue, Refers to an immobilized nucleic acid (LNA) or a mixture thereof. The term includes oligonucleotides consisting of natural nucleobases, sugars and internucleoside linkages (backbones), as well as oligonucleotides that function in the same way or that have improved specific function. It is. Fully or partially modified or substituted oligonucleotides can have several properties, such as cell membrane permeability, desirable resistance to intracellular and extracellular nucleolytic enzymes, and high affinity for target nucleic acids. Often preferred over the natural form because it has the desired specificity. LNA analogs exhibiting the above properties are particularly preferred.

  The term “unit” is understood as a monomer.

The term “at least 1” is an integer greater than or equal to 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, etc. Means.
The term “thio-LNA” means an immobilized nucleotide in which at least one of X or Y in Scheme 2 is selected from S or —CH ₂ —S—. Thio-LNA can be in both beta-D and alpha-L coordination.

The term “amino-LNA” means that at least one of X or Y in Scheme 2 is —N (H) —, —N (R) —, —CH ₂ —N (H) — or —CH ₂ —N (R ) _-Means a fixed nucleotide selected from R and selected from a hydrogen atom and a C _1-4 alkyl group. Amino-LNA can be in both beta-D and alpha-L coordination.

The term “oxy-LNA” means an immobilized nucleotide in which at least one of X or Y in Scheme 2 corresponds to O or —CH ₂ —O—. Oxy-LNA can be in both beta-D and alpha-L coordination.
The term “ena-LNA” refers to a fixed nucleotide where Y in Scheme 2 is —CH ₂ —O—.

The term “alpha-L-LNA” means a fixed nucleotide represented in Scheme 3.
The term “LNA derivative” means all fixed nucleotides except the beta-D-methylene LNA of Scheme 2, such as thio-LNA, amino-LNA, alpha-L-oxy-LNA and ena-LNA.

(Detailed description of the invention)
In the present invention, an oligomer compound, particularly an antisense oligonucleotide, used for regulating the function of a nucleic acid molecule encoding HIF-1α is used. Modulation is ultimately a change in the amount of HIF-1α produced. In one embodiment, this is accomplished by providing an antisense compound that specifically hybridizes to a nucleic acid encoding HIF-1α. Modulation is preferably inhibition of HIF-1α expression, which reduces the number of functional proteins produced. HIF-1 is involved in angiogenesis as well as involved in erythrocyte proliferation, cell proliferation, iron metabolism, glucose and energy metabolism, pH control, tissue invasion, apoptosis, multidrug resistance, cellular stress response or interstitial metabolism. concern.

  Antisense and other oligomeric compounds that regulate the expression of the target of the present invention are specific designs based on experimental or sequence information about the target and know-how of optimal oligomeric compound design for the desired target Identified by The sequence of these compounds is a preferred embodiment of the present invention. Similarly, sequence motifs (called hot spots) in targets where these preferred oligomeric compounds are complementary are preferred sites as targets.

  Preferred oligomeric compounds according to the invention are SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 97, 98, 99, 100, 101, 102, 103, 104, 1 A 5,106,107,108,109,110,111,112,113,114 or 115, indicating their sequence in Table 1 and Table 2. The oligomeric compounds according to the invention are powerful regulators of targets. This has been shown experimentally in vitro and in vivo. The in vitro inhibitory effects of the target using three different tumor cell lines are shown in Table 1 and FIGS. FIG. 9 shows the negative regulation (down regulation) of the target in vivo. Furthermore, oligomeric compounds have been shown to be potent inhibitors at lower concentrations compared to standard conditions for polynucleotide antisense with, for example, phosphorothioates. 5 and 6 show the inhibitory action when the concentration of the compound of the present invention is lowered to 5 nM. As shown in FIGS. 3 and 5, the inhibitory action of HIF-1α by the oligomeric compound of the present invention is the expression of vascular endothelial growth factor (VEGF) involved in angiogenesis and glucose transporter-1 ( It also inhibits the expression of GULT-1). Various designed oligomeric compounds targeting two sequence motifs (Table 2) were confirmed to be potent inhibitors of the target as shown in FIGS. Gene walking was performed using the oligomeric compounds from Table 1. And the effect of a powerful oligomeric compound is shown in FIG. All the experimental observations mentioned above show that the compounds according to the invention can be active compounds in pharmaceutical compositions.

Furthermore, the oligomeric compound according to the present invention inhibits HIF-1α in the state of normoxia and reduced oxygen pressure.
In one embodiment of the invention, the oligomeric compound comprises at least one unit of a compound designated LNA (Locked Nucleic Acid).
Representative citations for LNA monomers include WO 99/14226 and subsequent applications WO 00/56746, WO 00/56748, WO 00/66604, WO 00/125248, Bicyclic nucleoside analogs described in WO 02/28875, WO 02/094250 and International Application No. PCT / DK02 / 00488, all of which are incorporated herein by reference. A preferred LNA monomer structure is illustrated in Scheme 2.

X and Y are independently selected from the following groups: -O -, - S -, - N (H) -, - N (R) -, - CH 2 - or -CH- (double bond for a _{portion), - CH 2 -O -,} - CH 2 -S -, - CH 2 -N (H) -, - CH 2 -N (R) -, - CH 2 -CH 2 - or -CH _2- CH- (in the case of part of a double bond), -CH = CH-, wherein R is selected from a hydrogen atom and a _C1-4 alkyl group. Asymmetric groups can be seen in both configurations.

  In Scheme 2, four asymmetric centers are shown in one fixed configuration. However, what is included in the present invention is a compound represented by the general formula of Scheme 2, and the asymmetric center has a different configuration. Thus, each asymmetric center in Scheme 2 exists in either R or S configuration. The definition of R (rectus) and S (sinister) is described in “IUPAC 1974 Recommendations, Section E, Fundamental Stereochemistry”. These regulations are described in “Pure Appl. Chem. 45, 13-30 (1976)” and “Nomenclature of Organic Chemistry, Pergamon, New York, 1979”.

Z and Z ^* are independently selected from among internucleoside linkages, terminal groups or protecting groups.

The bonding mode between nucleosides is -OP (O) ₂ -O-, -OP (O, S) -O-, -OP (S) ₂ -O-, -SP (O ) ₂ -O-, -SP (O, S) -O-, -SP (S) ₂ -O-, -OP (O) ₂ -S-, -OP (O, S) -S-, -SP (O) ₂ -S-, -O-PO (R ^H ) -O-, -O-PO (OCH ₃ ) -O-, -O-PO (NR ^H ) _{-O -, - O-PO (} OCH 2 CH 2 S-R) -O -, - O-PO (BH 3) -O -, - O-PO (NHR H) -O -, - O-P ( O) ₂ —NR ^H —, —NR ^H —P (O) ₂ —O—, —NR ^H —CO—O—, —NR ^H —CO—NR ^H —, —O—CO—O—, —O _{^{-CO-NR H -, - NR}} H -CO-CH 2 -, - O-CH 2 -CO-NR H - ^{_{-O-CH 2 -CH 2 -NR H}} -, - CO-NR H -CH 2 -, - CH 2 -NR H -CO -, - O-CH 2 -CH 2 -S -, - S-CH 2 _{-CH 2 -O -, - S-} CH 2 -CH 2 -S -, - CH 2 -SO 2 -CH 2 -, - CH 2 -CO-NR H -, - O-CH 2 -CH 2 -NR ^H ₂ —CO—, —CH ₂ —NCH ₃ —O—CH ₂ — and the like may be used. Here, the ^RH group is selected from hydrogen and a C _1-4 alkyl group.

The end groups are hydrogen, azide group, halogen group, cyano group, nitro group, hydroxy group, Prot-O- group, Act-O- group, mercapto group, Prot-S- group, Act-S- group, C, respectively. _1-6 -alkylthio group, amino group, Prot-N (R ^H )-group, Act-N (R ^H )-group, mono- or di (C _1-6 -alkyl) amino group, optionally substituted C _1-6 - alkoxy group, an optionally substituted _{C 1-6} - alkyl group, optionally substituted _{C 2-6} - alkenyl group, an optionally substituted _{C 2-6} - alkenyloxy group , optionally C _2-6 substituted _- alkynyl group, an optionally substituted C _{2-6 -} alkynyloxy group, monophosphate group or a protected monophosphate group, monothiophosphate group or a protected Monothiophosphate groups, diphosphate ester groups or protected diphosphate ester groups, dithiophosphate ester groups or protected dithiophosphate ester groups, triphosphate ester groups or protected triphosphate ester groups, trithiophosphate ester groups Or independently selected from protected trithiophosphate groups. Examples of such protecting groups attached to phosphate ester residues include S-acetylthioethyl (SATE) or S-pivaloylthioethyl (t-butyl-SATE) groups, DNA intercalators, photochemical Active group, thermochemically active group, chelate group, reporter group, ligand, carboxyl group, sulfono group, hydroxymethyl group, Prot-O-CH ₂ -group, Act-O-CH ₂ -group, aminomethyl group , Prot-N (R ^H ) —CH ₂ — group, Act-N (R ^H ) —CH ₂ — group, carboxymethyl group, sulfonomethyl group. Here, Prot groups are protecting groups for —OH, —SH, —NH (R ^H ), Act groups are active groups for —OH, —SH, —NH (R ^H ), respectively, and R ^{The H} group is selected from hydrogen and a C _1-6 -alkyl group.

The protective group for the hydroxy substituent may be a substituted trityl group such as 4,4′-dimethoxytrityloxy group (DMT), 4-monomethoxytrityloxy group (MMT), and trityloxy group, which may be substituted 9 -(9-phenyl) xanthenyloxy group (pixyl), optionally substituted methoxytetrahydropyranyloxy group (mtp), trimethylsilyloxy group (TMS), triisopropylsilyloxy group (TIPS), tert-butyldimethyl Silyloxy groups (TBDMS), triethylsilyloxy groups, silyloxy groups such as phenyldimethylsilyloxy groups, tert-butyl ethers, acetals (including two hydroxy groups), acetyl groups or halogen-substituted acetyl groups (chloroacetyl) Oxy group, fluoroacetate Acyloxy groups such as isobutyryloxy group, pivaloyloxy group, benzoyloxy group, and substituted benzoyloxy group, methoxymethyloxy group (MOM), benzyloxys, or 2,6-dichlorobenzyloxy group (such as Substituted benzyloxys such as 2,6-Cl ₂ Bzl) are included. Alternatively, when Z or Z ^* are hydroxyl groups, they may be protected by attaching to the solid support via a bond if desired.

When Z or Z ^* is an amino group, examples of the protected amino group include fluorenylmethoxycarbonylamino group (Fmoc), tert-butyloxycarbonylamino group (BOC), trifluoroacetylamino group, allyloxycarbonylamino A group (alloc, AOC), a benzyloxycarbonylamino group (Z, Cbz), a substituted benzyloxycarbonylamino group such as a 2-chlorobenzyloxycarbonylamino group (2-ClZ), a monomethoxytritylamino group (MMT), A dimethoxytritylamino group (DMT), a phthaloylamino group, and a 9- (9-phenyl) xanthenylamino group (pixyl);

In the above embodiment, Act is an active group for —OH, —SH and —NH (R ^H ). In preferred embodiments, such active groups mediate coupling to other residues or monomers. After such a coupling is successful, the active group is converted to an internucleotide linkage. Such active groups are optionally substituted O-phosphoramidite groups, optionally substituted O-phosphate triester groups, optionally substituted O-phosphate diester groups, optionally substituted It is selected from a good H-phosphate group and an optionally substituted O-phosphate group.

The “phosphoramidite group” in this patent means a group represented by the formula —P (OR ^x ) —N (R ^y ) ₂ —, where the R ^x group is, for example, a methyl group, 2-cyanoethyl Group, or an optionally substituted alkyl group such as a benzyl group, and each R ^y group means an optionally substituted alkyl group such as an ethyl group, an isopropyl group, or the like, or —N (R ^y ) ₂ - group forms a morpholino group _{(-N (CH 2 CH 2)} 2) O). The R ^x group is preferably a 2-cyanoethyl group, and the two R ^y groups are preferably the same and represent an isopropyl group. Thus, a particularly suitable phosphoramidite group is the N, N-diisopropyl-O- (2-cyanoethyl) phosphoramidite group.

B constitutes a natural or non-natural nucleobase, adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 5-propynyl- From 6-fluorouracil, 5-methylthiazole uracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine, 2-chloro-6-aminopurine To be elected.

  A particularly preferred bicyclic structure is shown in Scheme 3 below:

When X is —O—, —S—, —NH— and N (R ^H ), Z and Z ^* are independently selected from among internucleotide bonds, terminal groups or protecting groups.
This internucleotide linkage is defined as -OP (O) ₂ -O-, -OP (O, S) -O-, -OP (S) ₂ -O-, -SP (O). ₂ -O-, -SP (O, S) -O-, -SP (S) ₂ -O-, -OP (O) ₂ -S-, -OP (O, S ) -S-, -SP (O) ₂ -S-, -O-PO (R ^H ) -O-, -OPO (OCH ₃ ) -O-, -O-PO (NR ^H ) -O- , —O—PO (OCH ₂ CH ₂ SR) —O—, —O—PO (BH ₃ ) —O—, —O—PO (NHR ^H ) —O—, —O—P (O) ₂ -NR ^H- , -NR ^H -P (O) ₂ -O-, -NR ^H -CO-O-, where the R ^H group is selected from hydrogen and a C _1-4 -alkyl group .
The end groups are hydrogen, azide group, halogen group, cyano group, nitro group, hydroxyl group, Prot-O- group, Act-O- group, mercapto group, Prot-S- group, Act-S- group, C _{1 -6} -alkylthio group, amino group, Prot-N (R ^H )-group, Act-N (R ^H )-group, mono- or di (C _1-6 -alkyl) amino group, optionally substituted C _1-6 -alkoxy group, optionally substituted C _1-6 -alkyl group, optionally substituted monophosphate ester group, monothiophosphate ester group, diphosphate ester group, dithiophosphate ester group, triphosphate ester Group and trithiophosphate group are independently selected. Each wherein Prot group, -OH group, a protecting group for -SH group, and -NH ^{(R H)} group, Act group each, -OH group, -SH group, and -NH ^{(R H)} group And the ^RH group is selected from hydrogen and a C _1-6 -alkyl group.

Examples of the protective group for the hydroxyl substituent include substituted trityl groups such as 4,4′-dimethoxytrityloxy group (DMT), 4-monomethoxytrityloxy group (MMT), and optionally substituted 9- (9-phenyl). ) Xanthenyloxy group (pixyl), optionally substituted methoxytetrahydropyranyloxy group (mtp), trimethylsilyloxy group (TMS), triisopropylsilyloxy group (TIPS), tert-butyldimethylsilyloxy group (TBDMS) ), Triethylsilyloxy groups, and silyloxy groups such as phenyldimethylsilyloxy groups, tert-butyl ethers, acetals (including two hydroxyl groups) and acyloxy groups such as acetyl groups. Alternatively, when Z or Z ^* are hydroxyl groups, they may be protected by attaching to the solid support via a bond if desired.

When Z or Z ^* is an amino group, examples of the protected amino group include fluorenylmethoxycarbonylamino group (Fmoc), tert-butyloxycarbonylamino group (BOC), trifluoroacetylamino group, allyloxycarbonylamino Groups (aloc, AOC), monomethoxytritylamino group (MMT), dimethoxytritylamino group (DMT), phthaloylamino group, and the like.

In the above embodiment, Act is an active group for —OH, —SH and —NH (R ^H ). In preferred embodiments, such active groups mediate coupling to other residues or monomers. After such a coupling is successful, the active group is converted to an internucleotide linkage. Such active groups include, for example, an optionally substituted O-phosphoramidite group, an optionally substituted O-phosphate triester group, an optionally substituted O-phosphate diester group, An H-phosphonate group which may be substituted and an O-phosphonate group which may be substituted.

As used herein, “phosphoramidite group” means the formula —P (OR ^x ) —N (R ^y ) ₂ —. Here, the R ^x group is an optionally substituted methyl group, an alkyl group such as a 2-cyanoethyl group, and each R ^y group is an optionally substituted alkyl group, and the R ^x group is preferred. Represents a 2-cyanoethyl group and the two R ^y groups are preferably identical and represent an isopropyl group. Thus, a particularly suitable group is the N, N-diisopropyl-O- (2-cyanoethyl) phosphoramidite group.

  B constitutes a natural or non-natural nucleobase, adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 6-aminopurine, It is selected from 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine.

Therapeutic Principles Those skilled in the art will understand that oligomeric compounds containing LNA can be used to treat HIF-1α related diseases according to many different principles, and that is therefore the intent of the present invention.

  For example, LNA oligomeric compounds can be designed as antisense inhibitors, which are single-stranded nucleic acids that block the production of disease-causing proteins at the mRNA level. In addition, they are designed as ribozymes or oligozymes that are antisense oligonucleotides, have a catalytic activity that degrades target mRNA in addition to the target binding domain (ribozyme), or an endonuclease that degrades target mRNA (RNase) It contains an external derivation sequence (EGS) that draws P) (oligozyme).

  Similarly, LNA oligomeric compounds can be designed as siRNA. They are small double-stranded RNA molecules that are used in cells and quiesce specific endogenous or exogenous genes by an “antisense-like” mechanism that is not yet well understood.

  LNA oligomeric compounds can be similarly designed as aptamers (and their variants called Spiegelmers). They are nucleic acids that bind to biological targets and block their function with high affinity and specificity by forming intermolecular hydrogen bonds from a three-dimensional structure. The LNA oligomer compound can be designed as a decoy. These are small double-stranded nucleic acids that inhibit the transcription of target genes by selectively interfering with the DNA binding sites of cellular transcription factors.

  Furthermore, LNA oligomeric compounds can be designed as chimeric plasts. They are small single-stranded nucleic acids that specifically pair and transform with a target gene sequence. Therefore, oligomeric compounds containing LNA using this principle are particularly useful for the treatment of HIF-1α related diseases caused by mutations in the HIF-1α gene.

  Finally, LNA oligomeric compounds can be designed as TFO (triple chain forming oligonucleotide). They are nucleic acids that block the production of proteins that bind to double-stranded DNA and cause disease at the stage of RNA transcription.

  It should be noted in the therapeutic principle of operating the oligonucleotide that the LNA oligomeric compounds according to the present invention preferably consist of about 8 to about 60 nucleobases, ie about 8 to about 60 linked nucleosides. Particularly preferred compounds are antisense nucleosides consisting of about 12 to about 30 nucleobases, most preferably antisense compounds consisting of about 12-20 nucleobases.

  Referring to the above principle for deriving the therapeutic action of the LNA oligomeric compound, the target of the present invention is the HIF-1α gene, mRNA or protein. In a most preferred embodiment, the LNA oligomeric compound is designed as an antisense inhibitor against HIF-1α pre-mRNA or HIF-1α mRNA. The oligonucleotide can hybridize to HIF-1α pre-mRNA or any site along the mRNA, eg, 5 'untranslated leader sequence, exon, intron and 3' untranslated end.

  In a preferred embodiment, the oligonucleotide hybridizes to human HIF-1α pre-mRNA or a portion comprising a translational inhibition site of mRNA. More preferably, the HIF-1α oligonucleotide comprises a CAT sequence, which is complementary to the HIF-1α pre-mRNA or the AUG start sequence of the mRNA. In another embodiment, the HIF-1α oligonucleotide hybridizes to a portion of a splice donor site of human HIF-1α pre-mRNA. In yet another embodiment, the HIF-1α oligonucleotide hybridizes with a portion of the splice acceptor site of human HIF-1α pre-mRNA. In other embodiments, the HIF-1α oligonucleotide hybridizes to a pre-mRNA of human HIF-1α or a portion involved in polyadenylation, transport or degradation of mRNA.

  Those skilled in the art will recognize that the preferred oligonucleotide is a portion of the HIF-1α pre-mRNA or part of the mRNA that hybridizes with a part that generally does not occur in transcription from unrelated genes, thus maintaining therapeutic specificity. You will understand that

  The oligomeric compounds of the invention are designed to be sufficiently complementary to the target so that there is an expected clinical response. That is, the oligomeric compound must bind with sufficient strength and specificity to the target in order to produce the expected effect. In one embodiment, the compound that modulates HIF-1α is designed to also modulate other specific nucleic acids that are not nucleic acids encoding HIF-1α.

  Preferably, the oligomeric compounds according to the present invention are designed to suppress intramolecular and intermolecular oligonucleotide hybridization.

  In many cases, confirmation of LNA oligomeric compounds that are effective in regulating HIF-1α activity in vivo or clinically is performed based on sequence information about the target gene. However, one of ordinary skill in the art will understand that such oligomeric compounds can also be confirmed by experimental testing. It is within the scope of the present invention that the HIF-1α oligomeric compound exhibits a response in clinical therapy, for example, with low sequence homology, some nucleotide modifications, or short lengths compared to preferred embodiments. include.

Antisense drug
In one embodiment of the invention, the oligomeric compound is a suitable antisense agent. The design of powerful and safe antisense drugs requires fine-tuning various parameters such as affinity / specificity, stability in body fluids, cellular incorporation, mode of action, pharmacokinetic properties And toxicity.

Affinity and specificity : LNA with oxymethylene 2′-O, 4′-C bonds (β-D-oxy-LNA) exhibits novel binding properties to DNA and RNA target sequences. Similarly, LNA derivatives such as amino-, thio- and α-L-oxy-LNA show novel affinity for complementary RNA and DNA, and in the case of thio-LNA, the affinity for RNA is β Even higher than -D-oxy-LNA.

  In addition to these remarkable hybridization properties, LNA monomers act in concert with other nucleic acid analogs such as DNA and RNA monomers and 2'-O alkylated RNA monomers when mixed. Thus, the oligonucleotides of the present invention can be composed solely of β-D-oxy-LNA monomers, or β-D-oxy-LNA and DNA, RNA or such as amino-, thio- and α-L-oxy- It can be composed of a combination with a current nucleic acid analog containing an LNA derivative such as LNA. The novel binding affinity of LNA for DNA or RNA target sequences, and its free mixing with DNA, RNA and the current range of nucleic acid analogs, develops effective and safe antisense compounds according to the present invention. In order to have an important significance.

    First, in one embodiment of the invention, the normal length of antisense oligos can be significantly shortened (from 20-25 mer to eg 12-15 mer) without reducing the pharmacologically required activity. Such a shortening significantly increases the specificity of the antisense compound for the RNA target, since the inherent specificity of the oligo is inversely related to its length. One embodiment of the present invention is to confirm in the target mRNA as short and unique as possible since the sequence of the human genome is available and its genetic code analysis is rapidly progressing.

  In another embodiment, the use of LNA to reduce the size of the oligo can greatly simplify the manufacturing process, resulting in antisense therapy becoming a market-competitive treatment for disease diversity. Providing a foundation for providing.

  In another embodiment, the novel affinity of LNA is used to substantially enhance the ability of antisense oligos to hybridize with target mRNAs in vivo, so as to identify active compounds. The time and labor required are significantly reduced compared to other chemical reactions.

  In another embodiment, the novel affinity of LNA is used to enhance the potency of antisense oligonucleotides, allowing the development of compounds with more advantageous therapeutic potential compared to current trials. .

  When designed as an antisense inhibitor, the oligonucleotides of the invention bind to the target nucleic acid and modulate the expression of its homologous protein. Preferably, the regulation is at least 10% or 20% inhibition of expression relative to the normal expression level, more preferably at least 30%, 40%, 50%, 60%, 70 compared to the normal expression level. %, 80%, or 90% inhibition of expression.

  Principally, the LNA oligonucleotide of the present invention contains, for example, natural DNA monomer, RNA monomer, N3′-P5 ′ phosphoramidite, 2′-F, 2′-O— in addition to β-D-oxy-LNA residue. Me, 2′-O-methoxyethyl (MOE), 2′-O- (3-aminopropyl) (AP), hexitol nucleic acid (HNA), 2′-F-arabino nucleic acid (2′-F-ANA) and And D-cyclohexenyl nucleoside (CeNA). Also, β-D-oxy-LNA modified oligonucleotides can include other LNA units in addition to or instead of the oxy-LNA group. In particular, additional preferred LNA units include thio-LNA or amino-LNA monomers having a configuration of D-β or L-α or combinations thereof, or ena-LNA. Typically, LNA modified oligonucleotides contain at least about 5, 10, 15, 20% LNA units based on the total nucleotides in the oligonucleotide, and more commonly all nucleotides in the oligonucleotide. On the basis of at least about 20, 25, 30, 40, 50, 60, 70, 80 or 90% of LNA units.

Stability in body fluids : In one embodiment of the present invention, standard DNA or RNA oligos are included to increase the stability of the resulting oligomeric compounds in body fluids, eg, resistance to nucleases (endonucleases and exonucleases). Incorporating LNA monomers into nucleotides is included. The degree of stability depends on the number of LNA monomers used, the position within the oligonucleotide, and the type of LNA monomer used. From a comparison with DNA and phosphorothioate, the order of the ability of oligonucleotide stabilization to degradative denaturation of nucleic acids can be determined as follows: DNA << phosphorothioate to oxy-LNA <α-L-LNA <amino-LNA <thio -LNA.

  Due to the fact that LNA is compatible with standard DNA synthesis and is free to mix with many current nucleic acid analogs, the nuclease resistance of LNA-oligomer compounds takes other analogs with higher nuclease stability. Or by utilizing nuclease resistant internucleoside linkages such as phosphoromonothioate, phosphorodithioate and methylphosphonate linkages.

Mechanism of action : Antisense compounds according to the present invention can elicit therapeutic effects through a variety of mechanisms and can combine several actions in the same compound. In one scenario, binding of an oligonucleotide to a target (pre-mRNA or mRNA) prevents the binding of other factors (proteins, other nucleic acids, etc.) necessary for the proper functioning of the target, Work to stop by physical obstacles. For example, antisense oligonucleotides bind to either pre-mRNA or mRNA sequence motifs that are important for the recognition and binding of processing factors involved in: splicing, polyadenylation, cell trafficking, in RNA Post-translational modification of 5'-end capping, translation, etc. In the case of pre-mRNA splicing, the result of the reaction between the oligonucleotide and its target is suppression of unwanted protein expression or generation of alternative spliced mRNA encoding the required protein, or both. In other embodiments, the binding of the oligonucleotide to its target causes a physical impediment to the ribosome mechanism, ie translational arrest, and prevents the translation process.

  In yet other embodiments, the binding of the oligonucleotide to the target interferes with the ability of the RNA to take on secondary and higher order structures that are important for proper functioning, i.e., cause structural interference. For example, oligonucleotides prevent the formation of stem and loop structures that play a vital role in various functions. Its function is, for example, to further stabilize RNA or to incorporate basic recognition motifs for various proteins.

  In yet another embodiment, the intracellular metabolic process is inhibited by collecting the intracellular enzymes that inactivate the target upon binding of the oligonucleotide and degrade the previous mRNA. For example, the oligonucleotide includes a nucleoside section that collects ribonuclease H (RNase H), which degrades the RNA portion of the DNA / RNA duplex. Similarly, an oligonucleotide can include a nucleoside segment that collects double-stranded RNAse, such as RNAse III, or can include an external derivation sequence (EGS) that collects an endogenous enzyme (RNaseP) that degrades the target mRNA. . Oligonucleotides can also include sequence motifs that exhibit RNAse catalytic activity, or moieties attached to the oligonucleotide, which negate the further metabolic activity of the target when approaching the target upon hybridization.

  It is known that β-D-oxy-LNA does not support RNase H activity. However, this can be changed by forming a chimeric oligonucleotide (called a gapmer) consisting of β-D-oxy-LNA and DNA according to the present invention. Gapmers are based on modified monomers flanked by 4-12 nt DNA with a central extension, or in particular 1-6 residues of β-D-oxy-LNA (flank) that are recognized and cleaved by RNaseH (gap). The flank may be constructed with LNA derivatives. There are other chimeric constructs according to the present invention, which act via a mechanism mediated by RNaseH. A headmer is defined by a continuous extension of β-D-oxy-LNA or an LNA derivative at the 5 ′ end, followed by a continuous extension of DNA or a 3′-terminal modification monomer that is recognized and cleaved by RNase H, It is defined by a continuous extension of DNA or a modified monomer at the 5′-end that RNaseH recognizes and cleaves, followed by a continuous extension of β-D-oxy-LNA or an LNA derivative at the 3′-end. Other chimeras according to the present invention are called mixmers, which are modified compositions that recognize and cleave DNA or RNaseH, and β-D-oxy-LNA and / or RNaseH binding and cleavage. Consists of LNA derivatives that may mediate. Since α-L-LNA supplements RNaseH activity to some extent, a small DNA gap or a modified monomer that RNaseH recognizes and cleaves is required for gapmer construction, and mixmer construction introduces more flexibility. FIG. 1 outlines various designs according to the present invention.

Pharmacokinetic properties The clinical effects of antisense oligonucleotides are highly dependent on pharmacokinetics such as absorption, distribution, cellular incorporation, metabolism and excretion. These parameters are then clearly derived from the underlying chemicals, size and three-dimensional structure of the oligonucleotide.

  As mentioned above, the LNAs described in the present invention are not single, but consist of several related components, all with surprising affinity for complementary DNA and RNA, albeit molecularly different. Show. Thus, the LNA chemical component is uniquely adapted to the development of oligos according to the present invention, which oligos have a high affinity for LNA that regulates the size of the active compound, or various that regulate the exact molecular composition of the active compound. It has controlled pharmacokinetic properties utilizing any of the LNA chemical components. In the latter case, for example, using amino-LNA rather than oxy-LNA changes the overall charge of the oligo, affecting the incorporation and distribution behavior. Similarly, the use of thio-LNA instead of oxy-LNA increases the lipophilicity of the oligonucleotide and affects the permeability of lipophilic barriers such as cell membranes.

  Modulation of the kinetic properties of LNA oligonucleotides according to the present invention is further effected by attachment of a wide variety of moieties. For example, the cell membrane permeability of an oligonucleotide is increased by attaching a lipid moiety such as, for example, a cholesterol moiety, thioether, aliphatic chain, phospholipid or polyamine to the oligonucleotide. Similarly, incorporation of LNA oligonucleotides into cells can be increased by coupling moieties to LNA oligonucleotides and acting on intramembrane molecules that mediate transport to the cytoplasm.

Pharmacological properties The pharmacological properties include improved biological stability, such as improved oligomer incorporation, increased resistance to degradation of the oligomer, and / or target sequences such as mRNA sequences and oligonucleotides. It is enhanced in accordance with the present invention by providing, for example, improved hybridization specificity and affinity.

The toxicity associated with toxicology antisense oligos basically two types, that is, class-related toxicity toxic and sequence-independent sequence-dependent, including the nucleotide sequence. Except for problems related to immunostimulation with native CpG sequence motifs, the most significant toxicity in the development of antisense oligonucleotides is not related to sequence, eg oligonucleotide chemistry and dosage, method, It relates to frequency and duration. The phosphorothioate class of oligonucleotides is particularly well characterized: complement activation, prolonged PPT (partial thromboplastin time), thrombocytopenia, hepatotoxicity (elevation of liver enzymes), cardiotoxicity, splenomegaly and reticulum It is known to induce many side effects such as endothelial hyperplasia.

  As previously mentioned, the LNA chemical moiety exhibits novel affinity, very high biological stability and the ability to modulate the exact molecular composition of the oligonucleotide. In one embodiment of the invention, compounds comprising LNA allow for the development of oligonucleotides that combine high titers and small amounts, if any, of phosphorothioate linkages, and thus are more effective than current antisense compounds. It is likely to show a high level of safety.

Production The oligo- and polynucleotides of the present invention can be produced using nucleic acid chemistry polymerization methods well known to those skilled in the art of organic chemistry. In general, a standard oligomerization cycle of the phosphoramidite method is used (SL Beaucage and RP Iyer, Tetrahedron, 49, 6123 (1993); SL Beaucage and RP Iyer, Tetrahedron, 48, 2223 (1992)), but, for example, chemical reactions of H-phosphonates and phosphorotriesters can also be used.

  For certain monomers of the present invention, long coupling times and / or repeated coupling with fresh reagents and / or more concentrated coupling reagents were used.

  Coupling with phosphoramidites was satisfactory with stepwise coupling yields exceeding 98%. Phosphate ester thiolation can be carried out by exchanging the usual oxidation reaction using, for example, iodo / pyridine / water, which is used for the synthesis of phosphorodiester oligomers, with an oxidation reaction using Beaucag reagent (commercially available product). It is also achieved with a sulfurization reagent. Phosphorothioate LNA oligomers were effectively synthesized with stepwise coupling yields greater than 98%.

  β-D-amino-LNA, β-D-thio-LNA oligonucleotides, α-L-LNA and β-D-methylamino-LNA oligonucleotides are also obtained by a stepwise coupling reaction using the phosphoramidite method. The synthesis was effective with a yield of 98% or more.

  Purification of the LNA oligomeric compounds was performed using a disposable reverse phase purification cartridge and / or reverse phase HPLC (high performance liquid chromatograph) and / or precipitation from ethanol or butanol. For confirming the purity of the synthesized oligonucleotide, gel electrophoresis, reverse phase HPLC, MALDI-MS (laser ionization mass spectrometry), and ESI-MS (electrospray ionization mass spectrometry) were used. In addition, solid supports on which the nucleobases and the protected LNA are immobilized if desired are particularly suitable as materials for synthesizing compounds containing LNA monomers at the 3 ′ end with LNA-containing oligomers. be interested. In this example, the solid support is preferably CPG, such as readily available (commercially available) CPG material or polystyrene, on the surface of which 3 ′ is functionalized and nucleobase and 5′-OH are desired. Protected LNAs are linked using conditions recommended by the vendor.

Indications The pharmaceutical composition according to the invention is used for the treatment of many different diseases. For example, in various human solid cancers and their metastatic cancers such as breast, colorectal, prostate, pancreas, brain, lung, ovary, gastrointestinal, head and neck, liver, bladder and cervix, HIF-1α is It has been found to be overexpressed (Zhong, H. et al., Cancer Research, 59, 5830-5835 (1999); Talks, KL et al., American Journal of Pathology, 157 (2), 411 -421 (2000)).

  The method of the invention is preferably used for the treatment or prevention of diseases caused by cancer, in particular for the treatment of cancers arising in the following tissues: lung, breast, large intestine, prostate, pancreas, liver, brain, testis, Stomach, small intestine, intestine, spinal cord, sinus, uterus, urinary tract and ovary.

  The invention further encompasses a method for the prevention or treatment of cancer comprising an amount of an HIF-1α modulating oligomeric compound that is effective in humans in need of treatment, including but not limited to high doses of oligomers. The invention further encompasses the use of HIF-1α modulating oligomeric compounds with short dosing periods. Normal non-cancerous cells divide with the frequency characteristics unique to that cell type. Unregulated cell division or proliferation is also evidence of cancerous cells, because when a cell is transformed to a cancerous state, it results in uncontrolled cell growth and decreased cell death. Examples of cancer types include, but are not limited to, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia (eg, acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia) , Multiple myeloma), colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell cancer, liver cancer, bile duct cancer, choriocarcinoma, cervical cancer, testicular tumor, lung cancer, bladder cancer, melanoma ( Malignant melanoma), head and neck cancer, brain tumor, unidentified cancer, neoplasm, cancer of the peripheral nervous system, cancer of the central nervous system, tumors (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, bone Sarcoma, chordoma, hemangiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ebbing tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma , Adenoma, sweat adenocarcinoma, sebaceous carcinoma, papillary carcinoma, breast Adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchogenic cancer, seminoma, fetal cancer, Wilms tumor, small cell lung cancer, epithelial cancer, glioma, astrocytoma, glioblastoma, craniopharynx , Ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma), heavy chain disease, metastasis, or disordered or abnormal Examples include diseases or diseases characterized by cell proliferation.

Pharmaceutical Compositions It is understood that the present invention also relates to pharmaceutical compositions that contain at least one antisense oligonucleotide composition of the invention as an active ingredient. The pharmaceutical composition according to the invention contains a pharmaceutical carrier if desired, and the pharmaceutical composition further contains an antisense compound, chemotherapeutic compound, anti-inflammatory compound, antiviral compound and / or immunomodulatory compound if desired. Understood.

Salts The oligomeric compounds encompassed by the present invention can be used as various pharmaceutically acceptable salts. As used herein, the term salts refers to salts that retain the necessary biological activity of the compounds identified in the present invention and that have minimal undesirable toxic effects. By way of non-limiting example, such salts are formed with organic amino acids and base addition salts are metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc. Or a cation derived from ammonia, N, N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium or ethylenediamine; or a combination of (a) and (b) in (c) such as zinc tannate. The

Prodrugs In one embodiment of the invention, the oligomeric compound can take the form of a prodrug. Oligonucleotides are fortunately negatively charged ions. Since cell membranes are lipophilic, the intracellular incorporation of oligonucleotides is low compared to neutral or lipophilic ones. This polarity disorder can be eliminated by the prodrug method (see, for example, Crooke, RM (1998) in Croke, ST, Antisense Research and Application. Springer-Verlag, Germany, vol. 131, pp. 103-140). )). In this method, the oligonucleotide is prepared in a protected form, so that the oligo when administered is neutral. After these protection groups are removed, the oligos are designed to be incorporated into the cells. Examples of such protecting groups include S-acetylthioethyl group (SATE) or S-pivaloylthioethyl group (t-butyl-SATE). These protecting groups are resistant to nucleases and are selectively removed intracellularly.

Conjugates In one embodiment of the invention, the oligomeric compound is linked to a ligand / conjugate. It is a method for increasing the intracellular incorporation of antisense oligonucleotides. This conjugation occurs at the terminal position of 5 ′ / 3′-OH, but in the case of coordination it occurs at the sugar and / or base site. In particular, the growth factor conjugated with the antisense oligonucleotide is preferably transferrin or folic acid. Transferrin-polylysine-oligonucleotide conjugates or folate-polylysine-oligonucleotide conjugates can be prepared for incorporation by cells expressing high levels of transferrin or folate receptors. Examples of other conjugates / ligands include a cholesteryl moiety, a double insert such as acridine and poly-L-lysine, one or more linked groups of nuclease resistance such as phosphoromonothioate, etc. And end capping.

  The preparation of transferrin complex as a carrier for oligonucleotide incorporation into cells has been reported by Wagner et al. (Wagner et al., Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990)). Intracellular transport of folate-macromolecular conjugates, including transfer of antisense oligonucleotides via folate receptor endocytosis, has been reported by Low et al. (Low et al., US Pat. No. 5,108, 921; see Leamon et al., Proc. Natl. Acad. Sci. USA, 88, 5572 (1991)).

Formulations The present invention includes formulations of one or more of the oligonucleotide compounds disclosed herein. Pharmaceutically acceptable binders and adjuvants are included as part of the formulated drug. Capsules, tablets, pills and the like contain, for example, the following compounds: microcrystalline cellulose, gum or gelatin as binder; starch or lactose as excipient; stearate as lubricant, various Sweet or flavoring agent. The capsule includes a liquid carrier such as a fatty oil in a prescription unit. Similarly, dragees or enteric solvents are part of the prescription unit. The oligonucleotide preparation can be an emulsion in which a micelle-like emulsion is formed with a pharmaceutically active ingredient and fat.

  The oligonucleotide of the present invention can be mixed with any material that does not impair the required action, or with a material that complements the required action. These can include other drugs, including other nucleoside compounds.

  Formulations for parenteral, subcutaneous, intradermal or topical administration include sterile diluents, buffers, isotonic control agents and antibacterial agents. The active compounds can be prepared using carriers that will protect against denaturation or rapid elimination from the body, including implants or microcapsules with controlled dissolution characteristics. Preferred carriers for intravenous administration are saline or phosphate buffered saline.

  Preferably, the oligomeric compound is a unit formulation, for example a unit containing a pharmaceutically acceptable carrier or diluent in a therapeutically effective amount sufficient to reach the patient without causing serious side effects It is a formulation.

Administration The pharmaceutical composition of the present invention can be administered in various ways, depending on whether it is local or systemic and where the site to be treated is. Methods of administration include: (a) oral, (b) transpulmonary, e.g. inhalation, powder or aerosol insufflation, including intratracheal or intranasal insufflation, (c) topical, epidermis, intradermal, Mucosal and rectal delivery, including the eye, vagina, or (d) parenteral, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion. Or intracranial, for example, intrathecal or intracerebral chamber administration. In one embodiment, the active oligo is administered as an intravenous (iv), intraperitoneal (ip), oral, topical or intravenous bolus, or directly to the target organ.

  Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, solutions and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like are necessary or desirable. Coated condoms and gloves are also useful. Preferred topical formulations include oligonucleotides of the invention in admixture with topical delivery agents such as lipids, liposomes, fatty acid esters, steroids, chelating agents and surfactants. Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous solvents, capsules, gel capsules, perfumes, tablets or mini-tablets. It is not limited to these. Compositions and formulations for parenteral, intrathecal or intracerebroventricular administration are sterile aqueous solutions containing buffers, diluents and other suitable additives including, but not limited to, Permeation enhancers, carrier compounds and pharmaceutically acceptable carriers or excipients are included.

Delivery Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions are produced from a variety of ingredients including, but not limited to, pre-prepared liquids, self-emulsifying solids and self-emulsifying semisolids. Delivery of drugs to tumor tissue is facilitated by carrier-mediated transport, which includes cationic liposomes, cyclodextrins, porphyrin derivatives, branched dendrimers, polyethyleneimine polymers, nanoparticles and microspheres (Dass, GR). , J. Pharm. Pharmacol, 54 (1), 3-27 (2002)), but is not limited thereto.

  The pharmaceutical preparation of the present invention can be easily provided in dosage unit units and can be prepared according to conventional methods well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with the pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

  The compositions of the present invention can be formulated into any dosage form such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels and suppositories. The compositions of the present invention can also be formulated as suspensions in water, non-aqueous or mixed solvents. Aqueous suspensions contain substances to further increase viscosity, such as sodium carboxymethyl cellulose, sorbitol and / or dextran. The suspension also contains a stabilizer.

Combinations The oligonucleotides of the present invention can be used to eliminate the effects of HIF-1α induced by acute hypoxia that occurs with androgen recovery therapy for prostate cancer.

  The oligonucleotides of the present invention can also be formulated with active agents such as aspirin, ibuprofen, sulfa drugs, antidiabetic drugs, antibacterial drugs or antibiotics.

  LNA-containing oligomeric compounds are useful for many therapeutic applications as described above. In general, the therapeutic methods of the invention comprise administering a therapeutically effective amount of an LNA modified oligonucleotide to a mammal, particularly a human.

  In certain embodiments, the present invention provides pharmaceutical compositions comprising (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents that are not antisense mechanisms. When chemotherapeutic agents are used with the compounds of the invention, individually (eg, mitramycin and oligonucleotide), sequentially (eg, mitramycin and oligonucleotide for a period of time, followed by another agent and oligonucleotide), Or in combination with one or more such chemotherapeutic agents, or in combination with radiation therapy. All chemotherapeutic agents known to the person skilled in the art are included as combination therapy with the compounds according to the invention.

  Although not limited thereto, anti-inflammatory agents, antiviral agents, and immunomodulators, including non-steroidal anti-inflammatory agents and corticosteroids, are similarly formulated in the compositions of the present invention. Two or more blended compounds are used together or sequentially.

  In other embodiments, the compositions of the present invention comprise one or more antisense compounds, particularly oligonucleotides, that target the first nucleic acid, and one or more additional antisenses that target the second nucleic acid. Contains compounds. Two or more blended compounds are used together or sequentially.

Dosage The dosage depends on the severity and sensitivity of the condition being treated and the course of treatment from days to months, or the period until recovery or disease disappears. Optimal dosing schedules are calculated from measurements of drug accumulation in the patient.

The optimal dosage will depend on the relative titer of the individual oligonucleotides. In general, it is estimated on the basis of an EC ₅₀ that is effective in vitro and in vivo experiments in animal models. In general, the dosage is from 0.01 μg to 1 g per kg of body weight, one or more times daily, weekly, monthly or yearly, once every 2 to 10 years, or several hours to several months Administered in a continuous infusion between. The repetition rate of administration is estimated based on the residence time and concentration measurement result of the drug in the body fluid or tissue. After successful treatment, it is desirable to have patients receive maintenance therapy to prevent recurrence of the disease.

Applications The LNA-containing oligomeric compounds of the present invention can be used as research reagents for diagnostics, therapeutics and prophylactics. In research, antisense oligonucleotides are used as specific inhibitors of HIF-1α gene synthesis in cells and in laboratory animals, thereby evaluating the utility of the target as a target for functional analysis or therapeutic intervention. It becomes easy. In diagnostics, antisense oligonucleotides are used to detect and quantify HIF-1α expression in cells and tissues by Northern blotting, hybridization or similar techniques. As a therapeutic agent, an animal or human suspected of having a disease or disorder that can be treated by modulating HIF-1α expression is treated by administration of an antisense compound according to the present invention. Further, a therapeutically or prophylactically effective amount of one or more of the present invention for treating humans or animals, particularly mice and rats, suspected or susceptible to a disease or condition associated with HIF-1α expression. Methods of administering an antisense compound or composition are provided.

Method The method of the present invention is preferably utilized for the treatment or prevention of a disease caused by one disease. One embodiment of the present invention is a method of inhibiting the expression of HIF-1α in a cell or tissue by contacting the cell or tissue with a compound of the present invention such that the expression of HIF-1α is inhibited. Become. Yet another embodiment comprises contacting the DNA or RNA of the gene with an oligomeric compound in a manner that regulates the expression of the gene involved in the disease. The oligomer compound is SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, It has a sequence comprising at least 8 nucleobase moieties of 108, 109, 110, 111, 112, 113, 114 or 115, thereby regulating gene expression. These compounds contain one or more LNA units. The compound hybridizes with the messenger RNA of the gene and inhibits its expression. Another embodiment is a method for treating a mammal suffering from or susceptible to a cancer disease, comprising administering to the mammal a therapeutically effective amount of an oligonucleotide that targets HIF-1α and comprises one or more LNA units. Become. The methods described above are common cancers such as pre-eclampsia, inflammatory bowel disease and Alzheimer's disease, such as primary and metastatic breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, other gastrointestinal cancer, lung cancer, uterus Cancer, ovarian cancer, brain tumor, head and neck cancer, cervical cancer, colon cancer, liver cancer, thymic cancer, kidney cancer, testicular tumor, gastric cancer, small intestine cancer, intestinal cancer, esophageal cancer, spinal cord cancer, sinus cancer, bladder cancer , And urinary tract cancers can be targeted. The described method regulates angiogenesis as well as involved in erythrocyte proliferation, cell proliferation, iron metabolism, glucose and energy metabolism, pH control, tissue invasion, apoptosis, multidrug resistance, cellular stress response or interstitial metabolism In the method, the cell comprises contacting the antisense compound of the present invention so that the cell is regulated.

  All documents mentioned here are incorporated herein by reference.

Examples The invention is described with specificity according to some preferred embodiments. Therefore, the following examples serve only to illustrate the invention and are not intended to be limiting.

Monomer Synthesis The preparation of the monomers shown in Scheme 2 where Y and X are —O— and Z and Z ^* are protected is described in detail in the literature. Koshkin et al., J. MoI. Org. Chem. 2001, 66, 8504-8512; Sorensen et al., J. MoI. Am. Chem. Soc. 2002, 124 (10), 2164-2176; Pedersen et al., Synthesis, 2002, 6, 802-809 and references cited therein. Here, the protecting groups for Z and Z ^* are oxy-N, N-diisopropyl-O- (2-cyanoethyl) phosphoramidite and dimethoxytrityloxy, respectively. Preparation of the monomer of Scheme 2 where X is —O— and Y is —S— and N (CH ₃ ) — is described by Rosenbohm et al., Org. Biomol. Chem. 2003, 1, 655-663.

LNA oligonucleotide synthesis All oligonucleotide synthesis is performed on a 1 μmole scale on the MOSS Expedite instrument platform. The synthesis procedure is carried out essentially as described in the instrument manual. The LNA monomer used is Koshin, A .; A. Et al. Org. Chem. 2001, 66, 8504-8512.

Preparation of LNA succinyl hemiester 5′-O-Dmt-3′-hydroxy-LNA monomer 500 mg), succinic anhydride (1.2 eq) and DMAP (1.2 eq) were dissolved in DCM (35 ml). The reaction was stirred overnight at room temperature. After extraction with NaH ₂ PO ₄ , 0.1M, pH 5.5 (2 times) and brine (1 time), the organic layer was further dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The hemiester derivative was obtained in 95% yield and was used without further purification.

Preparation of LNA-CPG resin The hemiester derivative prepared above (90 μmol) was dissolved in the minimum amount of DMF, DIEA and pyBOP (90 μmol) were added and mixed together for 1 minute. This pre-activated mixture was mixed with LCAA-CPG (500 kg, 80-120 mesh, 300 mg) and stirred in a manual synthesizer. After 1.5 hours at room temperature, the support was filtered off and washed with DMF, DCM and MeOH. After drying, the filling material was quantitatively determined to be 57 μmol / g.

5′-O-Dmt [A (bz), C (bz), G (ibu) and T] bound to phosphorothioate cycle CPG (solid-state support of porous glass beads, controlled pore glass) in dichloromethane solution. Deprotection was performed using 3 (v / v)% trichloroacetic acid. The resin was washed with acetonitrile. Coupling of phosphoramidites [A (bz), G (ibu), 5-methyl-C (bz) or T-β-cyanoethyl phosphoramidite] was achieved by a 0. 5′-O-Dmt protected amidite in acetonitrile. The activation was performed by using DCI (4,5-dicyanoimidazole) in acetonitrile (0.25M). Coupling was performed for 2 minutes. Thiolation was performed by using Beaucage reagent (0.05M in acetonitrile) and allowed to react for 3 minutes. The support is washed thoroughly with acetonitrile, followed by capping using unreacted acetic anhydride in THF (CAP A) and N-methylimidazole / pyridine / THF (1: 1: 8) (CAP B). The 5′-hydroxyl group was capped. The capping step was repeated and the cycle was terminated with an acetonitrile wash.

5′-O-Dmt [locA (bz), locC (bz), locG (ibu) or locT] bound to LNA cycle CPG (controlled pore glass) was deprotected using the same procedure as above. Coupling was performed using 5′-O-Dmt [locA (bz), locC (bz), locG (ibu) or locT] -β-cyanoethyl phosphoramidite (0.1 M in acetonitrile) and active The conversion was performed by DCI (0.25M in acetonitrile). Coupling was extended to 7 minutes. Capping was performed for 30 seconds using a solution of acetic anhydride in THF (CAP A) and a solution of N-methylimidazole / pyridine / THF (1: 1: 8) (CAP B). The phosphite triester is oxidized to the more stable phosphate triester by using a THF solution of I ₂ and pyridine for 30 seconds. The support is washed with acetonitrile and the capping step is repeated. The cycle was terminated with an acetonitrile wash. Abbreviations: Dmt: dimethoxytrityl, DCI: 4,5-dicyanoimidazole.

Oligonucleotide cleavage and deprotection The oligomer was cleaved from the support and the β-cyanoethyl protecting group was removed by treating the support with 35% NH ₄ OH for 1 hour at room temperature. The support was filtered off and the base protecting group was removed by heating at 65 ° C. for 4 hours. The oligo solution was then evaporated to dryness.

Oligonucleotide Purification Oligobodies are purified by (reverse phase) RP-HPLC or (anion exchange) AIE.
RP-HPLC:
Column: VYDAC ^™ , Catalog No. 218TP1010 (bydac)
Flow rate: 3 mL / min Buffer: A 0.1 M ammonium acetate, pH 7.6
B Acetonitrile gradient:
Time 0 10 18 22 23 28
B% 0 5 30 100 100 0
IE:
Column: Resource ^™ 15Q (Amersham Pharmacia Biotech)
Flow rate: 1.2 mL / min Buffer: A 0.1 M NaOH
B 0.1M NaOH, 2.0M NaCl
Gradient:
Time 0 1 27 28 32 33
B% 0 25 55 100 100 0
Abbreviations Dmt: Dimethoxytrityl DCI: 4,5-dicyanoimidazole DMAP: 4-dimethylaminopyridine DCM: dichloromethane DMF: dimethylformamide THF: tetrahydrofuran DIEA: N, N-diisopropylethylamine PyBOP: benzotriazol-1-yl-oxy-tris -Pyrrolidino-phosphonium hexafluorophosphate Bz: Benzoyl Ibu: Isobutyryl Beaucage: 3H-1,2-benzodithiol-3-one-1,1-dioxide A (bz), C (bz), G (ibu) or T: LNA-monomer (LNA: locked nucleic acid)

Cell Culture Antisense compounds and their effect on target nucleic acid expression can be measured in any of the various types of cells that have been subjected to a target nucleic acid or protein at a measurable level. This can be measured in a conventional manner using, for example, RT-PCR or Northern blot or Western blot analysis. The following cell types are provided for purposes of illustration, but other types can also be used routinely. Provided that the target is expressed in the selected cell type.

Cell lines were cultured under the appropriate conditions as described below and maintained at 37 ° C., 95-98% humidity and 5% CO ₂ . Cells were usually passaged 2-3 times a week.
U87-MG : The human glioblastoma cell line U87-MG was cultured in modified Eagle's medium (MEM) containing Earle's salt and 10% fetal calf serum (FCS).
U373 : The human glioblastoma cell line U373 was cultured in modified Eagle's medium (MEM) containing Yale salt and 10% fetal calf serum (FCS).
15PC3 : Human prostate cancer cell line 15PC3 is F. Contributed by Baas, Neurozinti Laboratories, AMC, Netherlands, and cultured in DMEM (Sigma) + 10% fetal calf serum (FBS) + GlutamaxI + gentamicin.

Anaerobic cell culture :
To monitor changes in HIF expression under hypoxic conditions, cells were cultured in an incubation bag (Merck) with Anaerocult (Merck) added to chemically bind O ₂ in 0.1-1.5. Incubated under anaerobic conditions with% O ₂ levels. Anaerobic conditions were obtained within 1-2 hours. Cells were anoxic for 6 or 18 hours.

Treatment with Antisense Oligonucleotides Cells (described above) were treated with oligonucleotides using the cationic liposome formulation LipofectAMINE 2000 (Gibco) as a transfection delivery vehicle. Cells were seeded in 100 mm × 20 mm cell culture Petri dishes (Corning) or 6-well plates (NUNC) and processed to 90% confluence. Oligo concentrations used ranged from 1 nM to 400 nM final concentration. The formulation of the oligo-lipid complex was performed according to the manufacturer's instructions using serum-free MEM and a final lipid concentration of 5 μg / ml in a total volume of 6 ml. The cells were incubated at 37 ° C. for 4 or 24 hours, and the treatment was stopped by removing the oligo-containing medium. Cells were washed and serum containing MEM was added. Following oligo treatment, cells were allowed to recover for 18 hours before 6 hours of hypoxia or directly 18 hours of hypoxia.

Total RNA Extraction Total RNA is isolated either using an RNeasy mini kit (eg, Qiagen catalog No. 74104) or using a Trizol reagent (eg, Life Technologies catalog No. 15596). It was. RNeasy is the preferred method for isolating RNA from cell lines, and Trizol is the preferred method for tissue samples. Total RNA was isolated from the cell line using the RNeasy mini kit (Qiagen) according to the protocol provided by the manufacturer. Tissue samples were homogenized using an UltraTurrax T8 homogenizer (eg, IKA Analysen Technik) and total RNA was isolated using the manufacturer-provided Trizol reagent protocol.

First-Strand cDNA Synthesis First-strand cDNA synthesis was performed using the OmniScript reverse transcriptase kit (Catalog # 205113, Qiagen) according to the manufacturer's instructions. For each sample, 0.5 μg of total RNA was adjusted to 12 μl each with RNase-free H ₂ O, poly (dT) _12-18 (2.5 μg / ml) (Life Technologies, GibcoBRL, Roskilde, DK) 2 μl, dNTPmix ( dNTPs 5 mM each) 2 μl, 10 × buffer RT 2 μl, RNAguard ^™ RnaseINHIBITOR (33.3 U / ml) (Catalog # 27-0816-01, Amersham Pharmacia Biotech, Hoersholm, DK) 1 μl, and OmniScr4 enzyme Mixed with 1 μl, then incubated at 37 ° C. for 60 minutes and heat inactivation of the enzyme at 93 ° C. for 5 minutes.

Analysis of antisense regulation of HIF-1α expression Antisense regulation of HIF-1α expression can be assayed by various known methods. For example, HIF-1α mRNA levels can be quantified by Northern blotting, antagonistic polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is currently preferred. RNA analysis can be performed on total cellular RNA or mRNA.

  RNA analysis methods such as RNA isolation methods and Northern blots are conventional techniques and are taught, for example, in Current Protocols in Molecular Biology, John Wiley and Sons.

  Real-time quantification (PCR) can be conveniently performed using a commercially available iQ multicolor real-time PCR detection system (available from BioRAD).

Real-time quantitative PCR analysis of Ha-ras mRNA levels Quantification of mRNA levels was determined by real-time quantitative PCR using the iQ multicolor real-time PCR detection system (BioRAD) according to the manufacturer's instructions.

  Real-time quantitative PCR is a well-known technique and is taught, for example, by Heid et al., Real time quantitative PCR, Genome Research (1996), 6: 986-994.

The platinum quantitative PCR supermix UDG2 × PCR master mix was obtained from Invitrogen catalog # 11730. Primers and TaqMan ^™ probes were obtained from MWG-Biotech AG, Ebersberg, Germany.

  Probes and primers for human HIF-1α are designed to hybridize to human Ha-ras sequences using publicly available sequence information (GenBank access number NM_00135, incorporated herein as SEQ ID NO: 1) It was done.

PCR primers for human HIF-1α are:
Forward primer: 5 ′ CTCATCCAAGAAGCCCTAACGTGTT 3 ′ (final concentration in assay: 0.9 μM) (SEQ ID NO: 116), reverse primer: 5 ′ GCTTTCCTGAGCATTCTGCAAAAGC 3 ′ (final concentration in assay: 0.9 μM) (SEQ ID NO: 117), and PCR probe 5′FAM-CCTCAGGAACTGTTAGTTCTTTGAACTCAAAGCGACA-TAMRA3 ′ (final concentration in the assay: 0.1 μM) (SEQ ID NO: 118).

  The amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control to normalize variations in sample preparation. The sample content of human GAPDH mRNA was quantified using the human GAPDH ABI Prism Pre-Developed TaqMan assay reagent (Applied Biosystems catalog No. 4310884E) according to the manufacturer's instructions.

  The following primers and probes were designed for quantification of mouse GAPDH mRNA: sense primer 5 'aaggctgtggggcaaggtcatc 3' (final concentration 0.3 μM), antisense primer 5 ' 'FAM-gaagctctactggcatgggcatggccttccgtgttc-TAMRA3' (final concentration 0.2 μM).

Real-time PCR
CDNA from first strand synthesis performed as described in Example 8 was diluted 2 to 20 times and analyzed by real-time quantitative PCR. Primers and probes were mixed with 2 × platinum quantitative PCR supermix UDG (Catalog # 11730, Invitrogen) and 3.3 μl of cDNA was added to a final volume of 25 μl. Each sample was analyzed in triplicate. A standard curve for the assay was generated by assaying 2-fold dilutions of cDNA prepared for material purified from cell lines expressing the relevant RNA. Sterile water was used instead of cDNA as a templateless control. PCR program: 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, followed by 40 cycles at 95 ° C., 15 seconds, 60 ° C., 1 minute.

  The relative amount of target mRNA sequence was determined from the threshold cycle calculated using iCycler iQ real-time detection system software.

Western blot analysis of HIF-1α protein levels HIF-1α protein levels are well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA, RIA (radioimmunoassay) or fluorescence activated cell sorting (FACS). It can be quantified by various methods. Antibodies directed to HIF-1α can be obtained from Upstate Biotechnologies (Lake Placid, USA), Novus Biologicals (Littleton, Colorado), various sources such as from Santa Cruz Biotechnology (Source: Santa Cruz, Calif.). Alternatively, it can be prepared by an ordinary antibody production method.

  To measure the effect of treatment with antisense oligonucleotides on HIF-1α, protein levels of HIF-1α in treated and untreated cells were measured using Western blotting.

  After treatment with oligonucleotides as described above, the cells are treated with a rubber policeman in ice-cold phosphate buffered saline (PBS) containing 0.37 mg / ml of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Obtained by scraping.

  The obtained cells were washed with 1 ml of PMSF-containing PBS as described above, and the cell pellet was stored frozen at −80 ° C.

  For protein extraction, frozen cell pellets were added to 3 volumes of ice-cold lysis buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 1% Non-idet P40 (Np-40), 0.1% SDS, 1% (w / v) sodium deoxycholate, 1 mM dithiothreitol (DTT), complete protein inhibitor cocktail (Boehringer Mannheim)]. Samples were sonicated with VibraCell 50 sonicator (Sonics & Materials Inc.) for 5-10 seconds and 2-3 times. The lysate was stored at −80 ° C. until use.

  The protein concentration of the protein lysate was measured using a BCA protein assay kit (Pierce) as per manufacturer's instructions.

SDS gel electrophoresis The protein sample prepared as described above was denatured at 70 ° C. for 10 minutes after thawing with ice.

  Samples are loaded onto a 1.0 mm 10% NuPageBis-Tris gel (NOVEX) and the gel is loaded with NuPage MES SDS Running Buffer or NuPage to achieve the desired separation of proteins using the XcellII minicell electrophoresis module (NOVEX). The electrophoresis was performed in either running buffer of MOPS SDS Running Buffer (both NOVEX).

  NuPage Antioxidant (NOVEX) was added to the running buffer to the inner chamber of the electrophoresis module to a final concentration of 2.5 μl / ml. As a size reference standard, SeeBluePlus2PrestainedStandard (Invitrogen) was loaded onto the gel. Electrophoresis was performed at 160 V for 2 hours.

After semi-dry blotting electrophoresis, the separated protein was transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry blotting. The membrane was equilibrated with NuPageTransferBuffer until it was adsorbed on the membrane. The blotting process was performed in Trans-blot SDS Semi-Dry transfer cells (BioRAD) according to the manufacturer's instructions. The membrane was stored at 4 ° C. until next use.

Immunodetection To detect the desired protein, membranes were incubated with polyclonal or monoclonal antibodies against the protein. The membrane was blocked by stirring for 1 hour with blocking buffer [2.5% skim milk powder and 5% BSA dissolved in TS buffer (150 mM NaCl, 10 mM Tris base, pH 7.4)]. The membrane was then washed 2 × 15 minutes with TS buffer at room temperature and incubated with the primary antibody overnight at 4 ° C. in TS-Tween 20 buffer containing 0.1% NaN ₃ . The following primary monoclonal antibodies and concentrations / dilutions were used: mouse anti-Glut1 (obtained from T. Ploug, The Panum Institute, Copenhagen) 1:20, mouse anti-HIF-1α (H72320, Transduction Laboratories) 1 μg / ml, mouse Anti-α-tubulin (T-9026, Sigma) 1: 10,000. Following incubation with the primary antibody, the membrane was washed with TS-Tween 20 buffer for 15 minutes, followed by two additional washes of 5 minutes each with stirring at room temperature. Subsequently, membranes were incubated for 1 hour at room temperature with a 1: 5000 dilution of secondary antibody, peroxidase-conjugated polyclonal goat anti-mouse immunoglobulin (P0447, DAKO A / S). The membrane was then washed with TS-Tween 20 buffer for 15 minutes, and further washed 3 times for 5 minutes at room temperature with stirring. After the final wash, the membranes were incubated with ECL ⁺ Plus (Amersham) for 5 minutes and then immediately scanned with STORM840 (Molecular Dynamics Inc.). The membrane was stripped by stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris base) pH 6.7 at 50 ° C. for 30 minutes with low agitation incubation. After washing with TS-Tween 20 buffer for 2 × 10 minutes at room temperature, the membrane was dried, sealed in a plastic bag and stored at 4 ° C. Protein expression levels were quantified for housekeeping protein expression using ImageQuant, ver5.0 software (Molecular Dynamics Inc.).

Antisense inhibition of human HIF-1α expression by oligonucleotides In accordance with the present invention, a series of oligonucleotides are used with published sequences (GenBank Access No. NM — 001530, incorporated herein as SEQ ID NO: 1 and FIG. 7). Designed to target different regions of human HIF-1α RNA. Oligonucleotides 16 nucleotides in length are shown in Table 1 and have SEQ ID NOs. Oligonucleotides are designed to be particularly effective when synthesized as antisense oligonucleotides, particularly using artificial nucleotides such as LNA or phosphorothioate. “Target site” refers to the first nucleotide number on the particular target sequence to which the oligonucleotide is bound. The compounds were analyzed for effects on HIF-1α protein by Western blot analysis as described herein in other examples.

  In this experiment, sequences that showed at least 20% inhibition of HIF-1α expression are preferred (see also FIGS. 1-9). Target sites for which these preferred sequences are complementary are referred to herein as “hot spots” and are therefore preferred sites to be targeted by the compounds of the present invention.

Antisense inhibition of HIF-1α by phosphorothioates, LNA containing oligonucleotides, or chimeric oligonucleotides having at least one LNA segment and at least one phosphorothioate segment In accordance with the present invention, a second series of antisense oligonucleotides was also synthesized. (Table 2). These series of compounds are fully modified phosphorothioate oligonucleotides, fully modified LNA oligonucleotides, or chimeric oligonucleotides of 16 nucleotides in length that target two different sites. A chimeric oligonucleotide is a region composed of phosphorothioate (PS), consisting of a region that is adjacent to an LNA segment at one or both sites, and is a “gapmer” (GM), “headmer” (WM5) (WM3). These regions consist of oxy LNA nucleotides. Some oligonucleotides are labeled with a fluorescent dye (FAM). Mismatched oligonucleotides were also designed (MM). All cytosines of oxy-LNA are methylated at the C5 site of the nucleobase. The compounds were analyzed for effects on HIF-1α protein levels by Western blot analysis as described in other examples. “Target site” indicates the first nucleotide number at the particular target sequence to which the oligonucleotide binds.

  Table 2. Inhibition of human HIF-1α protein levels by phosphorothioate oligonucleotides, LNA-containing oligonucleotides, or chimeric oligonucleotides having one or two LNA segments and one phosphorothioate segment (unless otherwise indicated, backbone binding is P = O) S: P = S bond, lower case: deoxynucleic acid, upper case: oxy LNA).

  As shown in Table 2 and FIGS. 1-6, most of SEQ ID NOs: 89-104 have demonstrated at least 20% inhibition of HIF-1α expression in this experiment and are therefore preferred. Target sites for which these preferred sequences are complementary are referred to herein as “hot spots” and are therefore preferred sites for targeting by the compounds of the present invention.

In vivo efficacy of oligomeric compounds targeting HIF-1α The effect of oligonucleotide treatment on the growth of tumor xenografts in nude mice can be measured using different tumor cell lines. Examples of such cell lines include human tumor cell lines U87 (glioblastoma), U373 (glioblastoma), 15PC3 (prostate cancer) and CPH54A (small cell lung cancer), and murine tumor cell lines There is B16 (melanoma).

Treatment of Subcutaneous Tumor Xenografts in Nude Mice Using LNA-Containing Oligo Tumor cells were implanted subcutaneously and then serially passaged by three consecutive transplants. A 1 mm tumor fragment was implanted subcutaneously into NMRI nude mice using a trocar needle. Separately, typically cancer cells were injected subcutaneously with 10 ⁶ cells suspended in matrigel (BD Bioscience) 300 [mu] l in the flank of NMRI nude mice.

Mice can be administered at various doses, with a maximum dose of 5 mg / kg / day, by intraperitoneal or subcutaneous injection of oligonucleotides, or up to 28 days using a subcutaneously implanted ALZET osmotic pump. Was treated by administration. Individual treatment of mice was initiated when the tumor volume reached 50 mm ³ . Treatment with PBS was initiated when the mean tumor volume of the control group reached 50 mm ³ . The experiment was terminated when either group of tumors reached the maximum allowable volume. The tumor size of all mice was measured daily by caliper measurement. The therapeutic effect was measured as tumor size and tumor growth rate. Oligonucleotide treated mice were disposed 24 hours after the last oligonucleotide injection.

  At the end of the treatment period, the mice were anesthetized and the tumor was removed and immediately frozen in liquid nitrogen for target analysis.

  Results: Mice bearing U373 xenograft tumors were treated with 5 mg / kg / day of Cur813 (SEQ ID NO: 97), intraperitoneally (ip) once daily for 7 days, or 100 μl / 10 g / day of PBS. Intraperitoneal (ip) administration once daily for 7 days. Each group, 5 mice were treated. Tumor assessment was performed as outlined above. The tumor growth curve is shown in FIG.

Comparison of tumor size (t test)
Day P value 4 0.0477
5 0.0156
6 0.0354
7 0.0461
Kaplan-Meier analysis final results: Tumor size 150 mm ³ :
Group No. Censored result Mean survival days PBS 5 0 5 5
Cur813 5 1 4 7
Log rank test for equivalence of survival distribution: P = 0.0138

Treatment of intracranial tumor xenografts in nude mice using LNA-containing oligos Tumor cells are implanted intracranially into NMRI nude mice and oligonucleotide treatment begins one week after transplantation. Mice are treated with various doses of oligonucleotides, intraperitoneal or subcutaneous injection at a maximum dose of 2 mg / kg, or by PBS administration. The number of treatments is determined by tumor growth in the control group. The experiment is terminated when either group of tumors reaches the maximum allowable size or death is assured in any group. The therapeutic effect is measured as the time to reach chronic nerve injury. Oligonucleotide treated mice are sacrificed 24 hours after the final oligonucleotide injection.

In vitro analysis: Inhibition of HIF-1α protein levels in human tumor cells grown in in vivo systemic treatment of antisense oligonucleotides Tumors were treated with lysis buffer [20 mM Tris C1 (pH 7.5); 2% Triton X-100; In 1/100 volume protease inhibitor cocktail set II (Calbiochem)] using a motor driven homogenizer at 4 ° C. 500 μl of lysis buffer was applied per 100 mg of tumor tissue. Tumor lysates were centrifuged at 13,000 G for 5 minutes at 4 ° C. to separate tissue debris. The protein concentration of the tumor extract was measured using the BCA protein assay reagent kit (Pierce, Rockford). Western blot analysis of target protein expression was performed as described in Example 8.

  The invention has been specifically described according to certain preferred embodiments thereof. Therefore, the following examples are merely illustrative of the present invention and are not intended to be limiting only.

FIG. 1 shows a Western blot of HIF-1α protein. Cells were treated with various 100 nM oligos for 4 hours. These cells were grown for 18 hours and then exposed to strict hypoxia for 6 hours. FIG. 2 shows a Western blot of HIF-1α protein. U87 cells were treated with 3 types of 200 nM oligos for 4 hours. These cells were exposed to strict hypoxia for 18 hours immediately after treatment. FIG. 3 shows a Western blot of HIF-1α, VEGF, Glut1 and tubulin proteins in U87 cells treated with oligo Cur0813. These cells were treated with 100 nM, 200 nM, 300 nM and 400 nM oligos for 24 hours. These cells were exposed to strict hypoxia for 18 hours immediately after treatment. FIG. 4 shows a Western blot of HIF-1α and tubulin protein in U87 cells treated with mismatched oligos (Cur0960 and Cur0961). These cells were treated with 100 nM, 200 nM, 300 nM and 400 nM oligos for 24 hours. These cells were exposed to strict hypoxia for 18 hours immediately after treatment. FIG. 5 shows a Western blot of HIF-1α, VEGF and tubulin protein in 15PC3 cells treated with oligo Cur813. These cells were treated with 125 nM, 25 nM, 5 nM and 1 nM oligos for 16 hours. These cells were exposed to strict hypoxia for 6 hours immediately after treatment. FIG. 6 shows a Western blot of HIF-1α and tubulin protein in 15PC3 cells treated with 5 nM of various oligos for 16 hours. These cells were exposed to strict hypoxia for 6 hours immediately after treatment. FIG. 7 shows a Western blot of HIF-1α and tubulin protein in U373 cells treated with 100 nM of various oligos for 6 hours. These cells were exposed to strict hypoxia for 20 hours immediately after treatment. FIG. 8 shows a Western blot of HIF-1α and tubulin protein in U373 cells treated with 100 nM of various oligos for 6 hours. These cells were exposed to strict hypoxia for 20 hours immediately after treatment. FIG. 9 shows the growth curve of xenograft U373 tumor when treated with phosphate buffered saline (PBS) or Cur813 at a dose of 5 mg / kg / day, intraperitoneally once daily for 7 days. Vertical bars indicate standard error. FIG. 10-1 is the gene sequence of human HIF-1α indicated using GenBank accession number NM_001530, which is incorporated herein as SEQ ID NO: 1. FIG. 10-2 is the gene sequence of human HIF-1α indicated using GenBank accession number NM_001530, which is incorporated herein as SEQ ID NO: 1, is there.

Claims (25)

An oligonucleotide compound that regulates the expression of HIF-1α, comprising the entire base sequence represented by SEQ ID NO: 55.   The oligonucleotide compound according to claim 1, wherein the compound specifically hybridizes with a nucleic acid encoding HIF-1α and inhibits expression of HIF-1α.   The oligonucleotide compound according to any one of claims 1 and 2, which is an antisense oligonucleotide.   The oligonucleotide compound of claim 3, wherein the antisense oligonucleotide consists of 16, 17, 18, 19, 20, or 21 nucleosides.   The oligonucleotide compound according to any one of claims 1 to 4, wherein the compound comprises at least one nucleoside analog.   6. The oligonucleotide compound according to any one of claims 1 to 5, wherein the compound comprises at least one locked nucleic acid (LNA) unit. The oligonucleotide compound according to claims 1 to 6, wherein the LNA unit has the structure of the general formula of Scheme 2:

Wherein, X and Y, -O -, - S -, - N (H) -, - N (R) -, - CH 2 -, - CH =, - CH 2 -O -, - CH 2 - _{S -, - CH 2 -N (} H) -, - CH 2 -N (R) -, - CH 2 CH 2 -, - CH 2 CH 2 =, and independently selected from -CH = CH-, Where R is hydrogen or C _1-4 -alkyl,
Z and Z ^* are independently selected from among internucleoside linkages, terminal groups or protecting groups; and B is a natural or non-natural nucleobase. The oligonucleotide compound of claims 1 to 7, wherein the at least one LNA unit has any formula according to Scheme 3 below:

Wherein X is independently selected from —O—, —S—, —NH— and N (R ^H );
R ^H is selected from H and a C _1-4 -alkyl group;
Z and Z ^* are independently selected from the group consisting of an internucleoside bond, a terminal group or a protecting group;
B constitutes a natural or non-natural nucleobase.   9. The oligonucleotide compound according to any one of claims 5 to 8, wherein the LNA unit is oxy LNA, thio LNA, amino LNA or a combination thereof of either D-β or L-α configuration.   From 2, 5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 LNA units 10. The oligonucleotide compound according to any one of 9 above.   11. The oligonucleotide compound of claim 10, comprising at least 4 LNA units. Adjacent nucleosides connected to each other by internucleoside linkages are -OP (O) ₂ -O-, -OP (O, S) -O-, -OP (S) _2- O-, -SP (O) ₂ -O-, -SP (O, S) -O-, -SP (S) ₂ -O-, -OP (O) _2- S-,- O—P (O, S) —S—, —S—P (O) ₂ —S—, —O—PO (R ^H ) —O—, —O—PO (OCH ₃ ) —O—, —O —PO (NR ^H ) —O—, —O—PO (OCH ₂ CH ₂ S—R) —O—, —O—PO (BH ₃ ) —O—, —O—PO (NHR ^H ) —O—. , —O—P (O) ₂ NR ^H —, —NR ^H —P (O) ₂ —O—, and —NR ^H —CO—O—, wherein R ^H is from hydrogen and C _1-4 alkyl. Selected from the group consisting of: Oligonucleotide compound according to any one of 3 to 11.   The oligonucleotide compound of claim 12, wherein the modified internucleoside linkage is a phosphorothioate linkage.   14. An oligonucleotide compound according to claims 1-13 comprising a continuous nucleoside region having the ability to collect RNAseH.   The oligonucleotide compound according to any one of claims 3 to 14, wherein the compound is a gapmer, a headmer or a tailmer.   The oligonucleotide compound according to claim 15, wherein the compound is a gapmer. From the 5 'end to the 3' end:
(I) a first region comprising 1 to 6 β-D-oxy LNA units;
(Ii) a second region, wherein the 5 ′ end of the second region is covalently linked to the 3 ′ end of the first region and comprises 4-12 deoxyribonucleosides or nucleoside analogs; A second region, and (iii) a third region, wherein the 5 ′ end of the third region is covalently linked to the 3 ′ end of the second region, and 1-6 β- The oligonucleotide compound of claim 16, comprising a third region comprising D-oxy LNA units.   The oligonucleotide compound according to any one of claims 1 to 17, which is covalently bound to a moiety that is not a nucleic acid or a nucleoside. An oligonucleotide compound targeting a nucleic acid molecule encoding HIF-1α, the oligonucleotide compound specifically hybridizing with a nucleic acid encoding HIF-1α and inhibiting the expression of HIF-1α And the compound comprises the entire base sequence shown in SEQ ID NO: 55.   A composition for inhibiting the expression of HIF-1α in a cell or tissue comprising the oligonucleotide compound according to any one of claims 1 to 19. A composition for regulating the expression of a disease-related gene, comprising an oligonucleotide compound having the entire base sequence represented by SEQ ID NO: 55.   24. The composition of claim 21, wherein the oligonucleotide compound comprises at least one LNA unit.   23. The composition of claim 21 or 22, wherein the oligonucleotide compound hybridizes with a gene messenger RNA and inhibits its expression.   A composition for treating a mammal suffering from or susceptible to cancer disease comprising a compound according to any one of claims 1-19.   25. The composition according to any one of claims 21 to 24, wherein the disease is a normal cancer selected from the group consisting of prostate cancer and brain tumor.

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